The objective of this project is to test the hypothesis that improved diagnostic techniques for breast cancer can be generated with agents recognizing GRSA proteins. Such agents comprise monoclonal antibodies and engineered single-chain variable region (scFv) forms of these antibodies. GRSA (Glycopeptide-Related Cell Surface Antigen) proteins are specific tumor products that result from the expression of the vasopressin (VP) gene. They are produced by seemingly all breast cancer and all ductal carcinoma in Situ (DCIS). Alternatively, VP gene expression and GRSA proteins are absent from normal breast tissue and from all breast fibrocystic conditions including atypical ductal hyperplasia (ADH). A confirmation of this distinction should provide a method for the early detection of initial and recurrent disease. Preliminary studies indicate GRSA proteins could represent a target for immunodiagnosis and for future therapeutic interventions. Therefore a principal focus of research over the funding period is to generate and evaluate ScFv GRSA-directed molecules from available antibodies for these purposes. Specific Aims are directed at: (i) assessing new immunohistochemical and RT-PCR diagnostic procedures for breast cancer and DCIS that feature monoclonal antibodies against GRSA proteins or primers for VPmRNA; (ii) manufacturing single-chain scFv diabodies and triabodies representing our monoclonal antibodies that recognize GRSA proteins on breast cancer; (iii) appraising these scFv molecules as useful clinical agents in vitro based on their ability to provide positive immunostaining of all breast carcinoma in an archival library, and to bind to cancer cells in culture, and; (iv)appraising these scFv molecules as useful clinical agents in vivo based on their ability to selectively target GRSA proteins on mouse tumor xenografts. Studies will employ RT-PCR, ligation, and cloning; ABC immunohistochemistry, Western analysis; flow cytometry; RIA; radiometric analysis of tissues; and gamma-camera scintigraphy. Expected outcomes of this research are the development of simple, sensitive, reliable, and universally available diagnostic methods that distinguish DCIS and breast cancer from atypical hyperplasia in tissue biopsies, and that can detect and localize recurrent disease in patients through radioimmunoimaging. These methods could be applicable to all patients with breast cancer or suspected breast cancer.